PROJECT SUMMARY Loss of tissue integrity and function is a unifying basis of disease, and the ability to regenerate lost or damaged tissue to its original form holds broad potential for human health. However, while some vertebrates such as newts can replace lost tissue perfectly, mammals repair injured tissue with scar, resulting in altered form and function. Why mammals have limited regenerative capacity is not known. A major barrier to addressing this limitation is our incomplete understanding of 1) how tissue is originally formed during development and 2) if and how these developmental programs can be resurrected in adults to drive regeneration. Currently, we lack mammalian models to explore these questions in parallel, and we lack a molecular handle to open up groundwork for systematic studies. Moreover, tools to dynamically trace how signals control specific cell behaviors that govern tissue formation are needed. In this grant, we address these challenges by examining mammalian skin, a model that uniquely exhibits regenerative wound healing. Here, full-thickness skin wounds on adult mice regenerate lost hair follicles through epithelial-mesenchymal interactions that chronicle embryonic skin development. This remarkable process is one of the few promising indications that mammals can achieve regeneration. Yet, how embryonic programs are reclaimed during wound healing is still unclear due to our fragmented understanding of how hair follicle formation is originally initiated during development and the lack of tools to trace molecular signals to specific cell behaviors that orchestrate hair follicle formation. In this grant, we examine how temporal activation of dermal Wnt/b-catenin prior to hair follicle initiation, coordinately regulates epithelial and dermal cell behaviors that mediate hair formation. We found that genetic modulation of dermal b-catenin at different time points prior to hair follicle initiation regulates hair follicle size. Based on this novel finding, we hypothesize that the timing of dermal Wnt/b-catenin signaling regulates hair follicle epithelial and dermal cell number in a tunable fashion during development and adult wound-induced hair follicle neogenesis. In Aim 1, we will use two-photon live imaging of mouse embryonic skin explants to coordinately delineate epithelial and dermal cell behaviors during hair follicle initiation and to dynamically track how temporal genetic loss or gain of dermal Wnt activity prior to hair follicle induction influences these cell behaviors. To identify molecular targets of dermal Wnt signaling, we will analyze transcriptomes of epithelial and dermal cell populations following ablation of dermal b-catenin at different time points. In Aim 2, we will begin to determine if dermal Wnt activation similarly regulates adult wound-induced hair follicle neogenesis. Specifically, we will define the spatiotemporal activity of dermal Wnt following wounding and analyze the size of neogenic hair follicles following genetic ablation of dermal Wnt at different times during wound healing. Success of this project will reveal mechanisms that drive hair follicle initiation and will set the stage for future studies that dynamically track the molecular and cellular mechanisms governing adult tissue regeneration.